Project Summary The continued development of protein quantification technologies is key to modern biology and medicine. Indeed, the ability to parallel process samples during quantitative analysis (multiplex) is essential for many of the proposed DBP applications. Sample multiplexing via chemical labeling offers numerous key advantages. First, parallel processing of samples in a single mass spectrometric analysis reduces the amount of material required from any one sample; this is particularly relevant for tissue-based work. Second, multiplexing greatly reduces sample preparation and instrument analysis time requirements, which is essential for the expanded application of proteomic analysis to translational medicine. Third, highly plexed analyses facilitate collection of biological replicate data, an obvious requisite for the field's advancement. Fourth, the ability to combine samples into a single analysis permits fewer MS experiments, which in turn allows for greater data overlap across all conditions. Finally, chemical labeling is compatible with biological tissues and fluids. We conclude the ability to analyze biological fluids and tissues is vital for the application of proteomics to translational medicine. In this TR&D project we aim to expand chemical labeling to an unprecedented 30-plex analysis, with the possibility to go much higher, by synthesis of new neutron-encoded chemical tags. Further, we will develop new technologies to boost MS/MS sampling overlap, since an MS/MS scan and identification is required to obtain quantitative data from isobaric tagging.